Skin model system

ABSTRACT

This invention relates to a skin model system that can be used as an in vitro test system or which can be used for therapeutic purposes. The skin model system comprises a three-dimensional, cross-linked matrix of insoluble collagen containing fibroblast cells therein, and stratified layers of differentiated epidermal cells supported thereon.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 08/441,850filed May 16, 1995, and issued May 26, 1998 as U.S. Pat. No. 5,755,814which is a continuation of application Ser. No. 08/352,979, filed Dec.9, 1994, which is now abandoned, which is a continuation of applicationSer. No. 08/032,373, filed Mar. 17, 1993, which is now abandoned.

FIELD OF THE INVENTION

This invention relates to artificial skin, its preparation, and its use.

BACKGROUND OF THE INVENTION

For some time, there has been a move to develop an artificial skin thatcan be used (1) for wound healing and the repair of ulcerated, burned orlacerated skin, and (2) as a model to test substances for irritation,toxicity, inflammation and pharmacology so as to reduce the number oftests using live animals. This latter application for artificial skin iscommonly referred to as an in vitro alternative to animal testing.

Berg and colleagues (U.S. Pat. Nos. 4,703,108 and 4,970,298, thedisclosures of which are herein incorporated by reference) havedeveloped a biocompatible, chemically crosslinked, three-dimensionalcollagen matrix as a substitute for the dermal layer of the skin whereit promotes fibroblast ingrowth and proliferation when implanted intoanimals (Doillon, et al., "Fibroblast-collagen sponge interactions andthe spatial deposition of newly synthesized collagen fibers in vitro andin vivo," J. Scanning Electron Microscopy, III:1313-1320 (1984). It hasalso been used to treat human Decubitus ulcers where it promotes healing(Silver, F. H., et al., "Collagenous materials enhance healing ofchronic skin ulcers," Biomedical Materials and Devices Research Society,110:371-376 (1989)). In vitro studies, this matrix has been used as amodel for examining the role of various matrix components on fibroblastingrowth (Doillion, et al., "Fibroblast growth on a porous collagensponge containing hyaluronic acid and fibronectin," Biomaterials8:195-200 (1987).

In this invention, this matrix is used as a support for humankeratinocyte growth and differentiation. The matrix described herein,containing keratinocytes and fibroblasts, is referred to herein as a"skin model system" or "SMS." Dollion, et al., in "Behavior offibroblasts and epidermal cells cultivated on analogues of extracellularmatrix," Biomaterials, 9:91-96 (1988), report on efforts to use the Bergcollagen matrix in attempts to manufacture artificial skin, but suchattempts were not successful. Epidermal cells on the surface of thematrix were neither differentiated nor in stratified layers.

An alternate collagen-based system has been developed by Bell et al.(Bell, E., et al., "The reconstitution of living skin," Journal ofInvestigative Dermatology, 81:2s-10s (1983); Bell, E., et al., "Recipesfor reconstituting skin," J. Biomechan. Eng., 113:113-119 (1991)) as adermal replacement called "living skin equivalent" or "LSE". The LSE ismanufactured by mixing living human fibroblasts with soluble rat tailcollagen under conditions where the collagen forms a gel (See, U.S. Pat.Nos. 4,485,096, 4,604,346, 4,8356,102, and Bell, E., et al., "Productionof a tissue-like structure by contraction of collagen lattices by humanfibroblasts of different proliferative potential in vitro," Proc. Natl.Acad. Sci. USA, 76:1274-1278 (1979)). During the five days of culture,the gel containing fibroblasts undergoes a contraction process where thecollagen volume is reduced to a small disc approximately 10% to 20% ofthe original volume depending on the concentration of collagen, the cellnumber and the composition of the growth medium. This contractedcollagen matrix is then used to support human keratinocyte growth.Although the Bell LSE is an advance over other previously knownartificial skin systems, it does suffer from disadvantages. Since themanufacture of the collagen matrix requires living fibroblasts, it isexpensive to manufacture and the matrix is not easily stored. The Bellcollagen matrix is not cross-linked and contracts with the addition ofthe fibroblasts, so it is difficult to manufacture the matrix in adesired shape and size. It is difficult to make large sizes of LSE; thematrix contracts substantially and large numbers of living cells arerequired. The Bell collagen matrix utilizes soluble collagen, which ismore difficult and expensive to extract than insoluble collagen. Stillfurther, since the Bell collagen matrix requires living cells, themanipulations to which it can be exposed are limited, e.g., it cannot beexposed to toxic conditions which might manipulate or favorably alterthe matrix structure hut which would kill the cells. In view of theselimitations, there remains a need for improved artificial skin systems.

SUMMARY OF THE INVENTION

It has now been found that a cross-linked matrix of insoluble collagencan be used as the matrix for preparing a skin model system mimickinghuman skin. This invention relates to such a skin model system,comprising a three-dimensional, cross-linked matrix of insolublecollagen containing fibroblast cells therein, and stratified layers ofdifferentiated epidermal cells supported thereon.

This invention further relates to a method for preparing such a skinmodel system comprising (a) providing a three-dimensional, cross-linkedcollagen matrix, (b) seeding said matrix with fibroblasts and culturingthe seeded matrix under conditions to allow ingrowth and proliferationof said fibroblasts, (c) seeding the surface of said matrix withepidermal cells in a manner to deter ingrowth of said epidermal cells,(d) culturing the seeded matrix for a first period of time underconditions to allow said epidermal cells to attach to said matrix andproliferate to form a monolayer and (e) culturing the seeded matrix fora second period of time under conditions to allow said epidermal cellsto differentiate.

Still further, this invention relates to methods for using the skinmodel system of this invention to determine the effect of an agent onhuman skin comprising contacting the skin model system with said agentand measuring the interaction of the skin model system and said agent.

Still further, this invention relates to methods for using the skinmodel system of this invention to treat wounds comprising transplantingsaid skin model system as a graft at the wound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning electron micrograph (SEM) of a cross-section ofunmodified, cross-linked collagen matrix of insoluble collagen. A 1000μbar is shown.

FIG. 2 is a graph of the streaming potential versus flow rate formatrices of this invention not containing polylysine (UMDNJ-DHT),matrices of this invention containing polylysine (UMDNJ-DHT-PL), and acommercially available collagen hemostatic sponge (INSTAT).

FIG. 3 is a graph of optical density of cell cultures on severaldifferent matrices, evidencing the ability of dermal fibroblasts toattach to these matrices. PLASTIC is polylysine-coated plastic tissueculture dish; INSTAT is a commercially available collagen sponge;INSTAT-C is a commercially available collagen sponge crosslinked withcyanamide; UMDNJ-C is a collagen matrix of this invention crosslinkedwith cyanamide; UMDNJ-DHT is a collagen matrix of this inventioncrosslinked by dehydrothermal treatment; UMDNJ-DHT-PL is a collagenmatrix of this invention crosslinked by dehydrothermal treatment andincorporating polylysine; UMDNJ-DHT-PA is a collagen matrix of thisinvention crosslinked by dehydrothermal treatment and incorporatingpolyaspartic acid. Cell density was determined by the MTT assay asdescribed in the examples.

FIG. 4 is a graph illustrating the ability of cells to grow on plastictissue culture dishes (PLASTIC) compared to their ability to grow on acommercially available collagen sponge (INSTAT), a cyanamide crosslinkedversion of the same matrix (INSTAT-CYANAMIDE), and a cyanamidecrosslinked version of the matrix described here (UMDNJ-CYANAMIDE).

FIG. 5 is a graph showing the effect of added polyamino acids on cellgrowth in collagen matrices. 3× cells were incubated either on tissueculture dishes (PLASTIC) or with one of several collagen matrices: thematrix described here crosslinked by a dehydrothermal technique(UMDNJ-DHT), the same matrix with polylysine (UMDNJ-DHT-PL), orpolyaspartic acid (UMDNJ-DHT-PA) incorporated during its manufacture.Cell density was determined by the MTT assay described in the examples.

FIGS. 6A-F are photomicrographs of cross-sections of human foreskin orof epidermal cell cultures grown on a collagen matrix. Magnification is400×, except were noted. FIG. 6A shows human foreskin. FIG. 6B shows theBell skin equivalent after ten days of culturing; FIG. 6C shows the sameafter 21 days of culture. FIG. 6D shows keratinocytes seeded on the "airside" of a cross-linked matrix of insoluble collagen (no polylysine orpolyaspartic acid). FIGS. 6E and 6F show the skin model system of thisinvention in which the matrix has been modified with polylysine.

DETAILED DESCRIPTION OF THE INVENTION

Skin is composed of both a dermal layer, consisting primarily of types Iand III collagen, proteoglycans, elastin and other matrixmacromolecules, and epidermal layers, consisting of epidermal cellscontaining keratin filaments undergoing progressive differentiation froma basal proliferating layer to a surface consisting of terminallydifferentiated, epidermal cells that protect the skin from theenvironment. The skin model system of this invention mimics thecomposition of normal skin. Fibroblast cells are grown within athree-dimensional matrix formed of cross-linked, insoluble collagen toform a dermal-type layer which supports stratified layers ofdifferentiated epidermal cells.

The collagen matrix utilized in this invention is based on insolublecollagen. "Insoluble collagen" refers to collagen which cannot bedissolved in aqueous acidic or alkaline or in any inorganic saltsolution without chemical modification. Preferred sources of theinsoluble collagen include hides, splits and other mammalian orreptilian coverings. More preferably, the collagen is derived from thecorium, the intermediate layer of a bovine hide between the grain andflesh sides. More generally, however, the collagen can be derived fromthe following typical sources: type I collagen: bovine, chicken and fishskin, bovine and chicken tendons and bovine and chicken bones includingfetal tissues; type II collagens: bovine articular cartilage, nasalseptum, sternal cartilage; and type III collagen, bovine and human aortaand skin.

The insoluble collagen is dispersed and swollen in a suitable liquidmedia, such as dilute hydrochloric acid, acetic acid or the like, havinga Ph between about 3.0 and 4.0. Generally, a weight ratio of insolublecollagen to dispersion agent of from about 1 to 15 is used to form thedispersion. This dispersion is poured into molds (generally, plastic ormetal trays) of the desired shape and size. For skin model systems, itis generally preferred that the thickness of the matrix be within about1 to 5 mm, preferably about 2-3 mm, so the size and shape of the moldcan be determined accordingly. The dispersion is solidified by freezingand is then lyophilized to form a three-dimensional, porous matrix.

Prior work has demonstrated that the pore size of the matrix isimportant to achieve optimal cell ingrowth. See U.S. Pat. No. 4,970,298,the disclosure of which is herein incorporated by reference. Generally,a matrix having a pore size of 50 to 250 microns, preferably in therange of 100±50 microns, containing channels is an ideal structure for acollagen-based material for cell ingrowth. (Pore size may be measuredfrom a photomicrograph using a ruler, averaging two measurements of apore taken at 90° angles from one another, and averaging suchmeasurements over a representative number of pores.) The optiumconditions for forming a matrix having these characteristics are: (1) toavoid excessive blending and obtain a well dispersed mixture of largecollagen fibers, (2) to disperse the collagen in a liquid media having aPh of about 3.0 to about 4.0, (3) to freeze the collagen dispersion tofrom about -30° C. to about -50° C. in an ethanol bath, and (4) to keepthe ethanol bath in direct contact with the plastic or metal tray toavoid any air gap. After freezing, lyophilization is generally carriedout under conditions of a sample temperature of about 0 to 20° C. and avacuum below about 200 mTorr Hg.

As noted in U.S. Pat. No. 4,970,298, the upper surface of the sponge indirect contact with the atmosphere during the freeze-drying process,called the "air side", is found to have a collapsed form or a sheet-likestructure in almost all cases. The other side of the sponge, in directcontact with the tray, called the "pan side", is found to have a moreopen, delicate structure. The average pore size on the pan side tends tobe significantly smaller (e.g., generally at least about 100μ smaller)than that of the air side. Not reported in U.S. Pat. No. 4,970,298, butdescribed hereinbelow, is the importance of seeding the epidermal cellson the "pan side" of the sponge rather than the "air side" to achievethe optimal skin model system.

Prior to solidifying the collagen dispersion, other agents may beincorporated into the dispersion. For example, polycationic polymerssuch as polylysine or polyaspartic acid may be incorporated to improvecell growth. Carrier compounds such as collagen types IV and V,fibronectin, laminin, hyaluronic acid, proteoglycans, epidermal growthfactor, platelet derived growth factor, angiogenesis factor, antibiotic,antifungal agent, spermicidal agent, hormone, enzyme and enzymeinhibitor.

Following lyophilization, the collagen matrix is cross-linked.Preferably, cross-linking is carried out by a dehydrothermal treatment.Suitable conditions include subjecting the matrix to temperatures offrom about 50° C. to about 200° C. at a vacuum of 50 millitorr or lessfor a period of time from about 2 to 96 hours. However, the collagen mayalso be cross-linked by chemical agents (or a combination ofdehydrothermal treatment and chemical agents) such as carbodiimide orsuccinimydyl ester/carbodiimide.

Examples of the carbodiimides include cyanamid and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. Examplesof bifunctional succinimidyl active esters include bifunctionalN-hydroxysuccinimide, 3,3'-dithio(sulfosuccin-imidyl)proprionate andbis(sulfosuccinimidyl)suberate. When using a carbodiimide cross-linkingagent, the collagen-based matrix is immersed in a carbodiimide solutionat a concentration of from about 0.1 to 10% (W.V) maintained at atemperature of from about 2° C. to 40° C. and at a Ph of between 2 to 11for a period of time of from about 2 to 96 hours. When using asuccinimidyl active ester crosslinking agent, the collagen-based spongeor sheet is immersed in a solution thereof at a concentration of fromabout 0.1 to about 15.0% (W/V) maintained at a temperature of from about2° C. to 40° C. for a period of time of from about 2 to 96 hours. Thecollagen-based sheet is placed in a solution containing 0.1 to about 15%(W/V) of N-hydroxysuccinimide and carbodiimide at a Ph between 2 to 11for a period of time between 2 to 96 hours at a temperature of fromabout 2° C. to 40° C. The thus-treated intermediate collagen-basedmatrix is exhaustively washed to remove excess cross-linking agent.

Unlike prior art collagen matrices prepared from collagen and livingcells, the collagen matrices of this invention can be stored for periodsof time in a dry state. For example, experience has suggested thatsterilized matrices can be stored under dry and sealed conditions inexcess of two years. Prior to storage or use, it is generally preferredto sterilize the matrices, e.g., with gamma irradiation. Prior toseeding the matrices with living cells, the matrices are preferablytreated (e.g., soaked overnight in DMEM supplemented with 10% calfserum) to remove any residual acid.

The next step in preparing the skin model system described herein isseeding the collagen matrix with fibroblasts. Fibroblasts will bedispersed throughout the collagen matrix to simulate the dermal layer ofhumans and other mammals. Although the type of fibroblast utilized isnot critical, it is preferred to use dermal fibroblasts as they willdeposit the appropriate types of collagen and other dermal components.The fibroblasts may be of human or animal origin. They may becommercially obtained or may be cultured from a patient biopsy.Fibroblast-like cells may also be used. Other cells may also be culturedin the collagen matrix, including but not limited to endothelial cells,pericytes, macrophages, monocytes, lymphocytes, plasma cells andadipocytes.

The fibroblasts are generally inoculated onto the matrix at a density ofabout 0.2 to 1×10⁶ cells per 2×2 centimeter of matrix. Preferably, thefibroblasts are inoculated onto the "air side" of the matrix as thegreater pore size on this side encourage ingrowth of the fibroblastsinto the matrix. However, it should also be possible to seed the "panside" of the matrix. As mentioned above, pore sizes in the range ofabout 100±50μ have been found to be ideal for cell ingrowth. The seededmatrix is cultured for a period of time and under conditions to allowthe cells to grow and modify the collagen matrix so that it will supportthe growth of epidermal cells. During this period, the fibroblastssubstantially fill the spaces in the collagen matrix, generally, aperiod of from about one to fifteen days, preferably about five days.Suitable culture conditions would be known to those skilled in the artand would be conditions conducive to the proliferation of thefibroblasts. Such conditions include immersing the seeded matrix in DMEM(Dulbeco's Modified Eagle's Medium) supplemented with 5% FBS (fetalbovine serum) or 10% bovine serum for five to seven days.

The next step involves seeding the matrix with epidermal cells. Theseepidermal cells must be keratinocytes, although other cells may be usedin conjunction with the keratinocytes (e.g., melanocytes). Epidermalcells are generally inoculated onto the matrix at a density of about100,000 to about 500,000 cells per cm², although lesser or greateramounts could be used. The epidermal cells must be seeded onto the "panside" of the matrix. It has been found that if the cells are seeded ontothe "air side", with its open, large pore structure, the cells migrateinto the matrix to produce swirls and pockets of epidermal cells.Applying epidermal cells to the pan side, that side with the smallestpore structure, improved epidermal cell growth and proliferation byminimizing ingrowth of the epidermal cells into the matrix.

The matrix seeded with epidermal cells is cultured for a first period oftime and under conditions suitable to allow the epidermal cells toattach and form a monolayer. Generally, this may be accomplished bysubmerging the seeded matrix in an epidermal growth media forapproximately one week at 37° C. and 7% CO₂. A suitable epidermal growthmedia ("E-media") contains: 3:1 high glucose DMEM supplemented withHam's F12 Nutrient Mixture (Gibco), 5×10⁻¹⁰ M cholera toxin(Schwarz-Mann Biotech), 2 μg/ml hydrocortisone, 25 μg/ml insulin, 25μg/ml transferrin, and 1×10⁻¹⁰ M triiodothyronine (all from SigmaChemical Company).

Once the epidermal cells have proliferated to form a monolayer, thematrix is cultured for a second period of time under conditions to allowthe epidermal cells to differentiate. The culture or matrix is raised sothat the surface seeded with epidermal cells is exposed to theatmosphere, e.g., by raising the matrix to the liquid/air interface inthe culture. The matrix continues to be fed with the epidermal growthmedia and held until the epidermal cells have differentiated as desired.A period of twenty-one days has been found to be sufficient, althoughshorter or longer periods may be utilized.

Tests indicate that the epidermal cells grown on the collagen matrix asdescribed above form stratified layers similar to skin with theepidermal cells differentiating as they approach the surface. Thoseskilled in the art would know how to determine whether a stratifiedlayer of cells is present. Stratified layers of epidermal cells arefound where several layers of cells are piled on top of one another asopposed to being in a single monolayer (or to simply filling in holes inthe collagen matrix). Generally, the stratified layers formed in thisinvention will comprise a layer of three or more, preferably five ormore, cell thicknesses.

Those skilled in the art would also know how to determine whetherepidermal cells have differentiated. Cells that have differentiated showa change in morphology (shape and appearance) and often secrete certainproteins. Changes in morphology include (1) the presence of granules inthe granular layer (or a layer in the SMS approximating the granularlayer), (2) the flattening of the cells and (3) the loss of cell nuclei.The presence of any one or more of these changes in morphology indicatesthat the cells have differentiated or are differentiating.

When epidermal cells differentiate, they also start to make certainproteins. For the purpose of this invention, the presence of any one ormore of those proteins in the epidermal cells is evidence that the cellshave differentiated or are differentiating. Those proteins includeinvolucrin, filaggrin, keratin K1 and loricrin.

Involucrin is a cell envelope protein present in all but the basallayers of normal skin and is one of the earlier proteins produced forthe formation of the corneocyte envelope in human skin. The corneocyteenvelope is a rigid alkaline-resistant structure produced only bydifferentiating keratinocytes and not by basal keratincoytes.

Filaggrin is a protein produced in the suprabasal layer as profilaggrinwhich is processed to filaggrin. In human skin, it is found in the upperstratum spinosum and granulosum. Filaggrin is generally not found in thestratum corneum, possibly because it is degraded to amino acids in thatlayer. Filaggrin is involved in the preparation of keratohyalin granulesformed in differentiating cells of the epidermis. Only differentiatingcells would produce filaggrin.

Keratins are a family of proteins whose members are expressed as afunction of the differentiation state of the epidermal cells. Thekeratin K1 (having a molecular weight of 67 kDa), is the keratin presentin the most highly differentiated keratinocytes. In normal skin, K1 islocalized to the upper stratum spinosum and granular layers.

Loricrin is a major cornified envelope precursor, which is one of thelatest envelope proteins to be expressed in normal skin. When using anantibody specific to the epitopes on the N-terminal portion of loricrin,normal foreskin has been found to stain specifically from the upperstratum spinosum to the stratum corneum.

The presence of these proteins in the skin model system can bedetermined by standard immunohistochemical techniques described in moredetail in the Examples. Tests show that the amounts and distribution ofeach of these proteins in the skin model system of this invention mimictheir distribution in normal neonatal skin. The only exception to thispattern was that reduced amounts of loricrin were determined in the skinmodel system matrix described herein compared to normal skin.

The skin model system described herein should have a variety of uses.Chemicals such as drugs, cosmetics, pesticides and food additives mustoften be tested for skin irritation, for toxicity and/or for efficacy.Currently, much of this testing is carried out on laboratory animals.The skin model system of this invention, however, closely resembleshuman skin and can be used not only to spare laboratory animals but alsoto more accurately gauge the effect of a chemical on human skin. Oneaspect of this invention, therefore, relates to a method of determiningthe effect of a chemical or other agent on human skin comprisingcontacting a skin model system of this invention with said chemical oragent and measuring the interaction of the skin model system and saidchemical or agent. The term "agent" is intended to encompass not onlysubstances but conditions such as light, heat, etc. Such interactionscould include, but are not limited to, the release of one or moresubstances by the skin model system, an effect on metabolism or cellproliferation or differentiation of the cells, or the reorganization ofthe cells of the system. The extent to which the chemical or agent islikely to affect human skin is determined by the extent of any suchinteraction with the skin model system. In this way, the potentialtoxicity or potential for irritation of a chemical or other agent may bedetermined, as may the potential pharmacological efficacy of a chemicalor other agent be determined.

Other utilities for the skin model system of this invention include itsuse as a model for studying skin diseases and developing new treatmentsfor skin ailments. For example, one could form the skin model system ofthis invention using cell lines from patients with a certain disease tolearn more about that disease and to study and evaluate the efficacy oftreatments for it. More specifically, one aspect of this inventionrelates to a method for determining the efficacy of a treatment for askin condition comprising (a) providing the skin model system of thisinvention wherein diseased cells typical of said condition have beenused to produce said system, (b) exposing said skin model system to saidtreatment, and (c) monitoring any change in said skin model system.

The skin model system of this invention may also be used for treatingpatients suffering from a wound to the skin, for example, burn patients.The skin model system may be applied to the wound, for example, bytransplantation or grafting.

This invention is further illustrated by the following examples, whichare provided for purposes of illustration only. These examples are notintended to limit the scope of this invention.

EXAMPLE 1

Collagen Matrix Formation

Three-dimensional collagenous matrices were prepared from collagen(Devro, Inc.; microcut) which was isolated from the corium layer of thebovine hide by shredding and washing in various solvents in order toobtain a slurry of insoluble collagen particles in water. See, forexample, U.S. Pat. No. 4,703,108, U.S. Pat. No. 4,970,298 and Jain, M.K., et al., "Material Properties of Living Soft Tissue Composites," J.Biomed. Mat. Res., 22:311-326 (1988), the disclosures of which areherein incorporated by reference. Since collagen swells when exposed toacid, the slurry was prepared by adding 6 gm collagen to 100 ml 0.001 NHcl, Ph 3, and allowed to swell. The slurry was then blended to producea smooth dispersion. (In some preparations, 30 mg polylysine were addedper 100 ml of dispersion corresponding to 0.5% polylysine per collagenwt/wt, as described below in Example 2.) The dispersion of collagen inacid (250 ml) was then poured into molds of teflon lined pans 8" by 8"to a depth of 3-4 millimeters. The molds had flat bottoms and were opento the air on top. The dispersions were frozen at -20° C. andlyophilized to form a matrix. The matrix was then crosslinked using adehydrothermal treatment (110° C. in a vacuum of less than 10 m torr).The collagen matrices were cut with a cork borer to a diameter of 1.5 cmor with a razor blade to squares of approximately 2 cm by 2 cm. Thematrices were resealed in plastic and sterilized with gamma irradiationusing 2.5 m rad of ⁶⁰ Co (Isomedix, Inc.) and stored in a dry state(FIG. 1).

The pore structure of the matrices produced by freezing and lyophilizingvaried from approximately 50 to 200μ. The pore structure at the surfaceof the matrix was more open for the air side of the matrix and moreclosed for the side of the matrix in contact with the mold.

EXAMPLE 2

Preparation of Modified Collagen Matrices

In attempt to change the surface charge on the matrices to improve cellattachment and growth, matrices were modified by the incorporation of0.5% wt per wt polylysine or polyaspartic acid per collagen. Thepolylysine or polyaspartic acid was added to the collagen in the aciddispersion for incorporation into the sponge prior to pouring into themold and freeze drying. In order to determine if the surface charge waschanged, the matrices were examined by measuring their streamingpotential. Streaming potentials of matrices were measured with ahorizontal streaming potential chamber. See Walsh, W. R., et al.,"Electrokinetic Effects on Matrix Fibroblast Interactions," BRAGS(1992). The electrical potentials in 0.145 M, pH 7.3, sodium veronalbuffer were measured using silver/silver chloride electrodes and aKeithley 642 Electrometer and x-t recorder. Collagen sponges wereequilibrated in the streaming potential chamber for 30 minutes followedby 5 minutes at a flow rate of 1 ml/min. Streaming potentials at severalflow rates, corresponding to different pressures, were determined after30 seconds of flow at a given pressure when the signal was stable asindicated on x-t recorder. The slope of the flow rate versus streamingpotential is inversely related to the zeta potential. The zeta potentialis defined as the average potential difference between the bulksolution, considered to be zero potential, and the surface of shear(near the matrix).

Results are preserved in FIG. 2 as a graph of the streaming potentialversus flow rate for the matrix of this invention not containingpolylysine (UMDNJ-DHT), matrices of this invention containing polylysine(UMDNJ-DHT-PL), and a commercially available collagen hemostatic sponge(INSTAT) (INSTAT™ sponges are a product of Johnson & Johnson Co.) Thestreaming potentials for all collagen matrices tested were negative atpH 7.3 in 0.145 M buffer. Since the slope of the flow rate versusstreaming potential is inversely related to the zeta potential with theelectrode polarity used in the streaming potential chamber, the matricesall had positive zeta potentials. The zeta potential was positive forthe matrices without polylysine and more positive for the polylysinematrices (FIG. 2). The streaming potential increased as the flow rateincreased (applied pressure increased), as predicted by electrokinetictheory. (Lyklema, J., et al., J. Colloid Sci., 16:501-512 (1961).

EXAMPLE 3

Cell Attachment and Growth Curve Assays in Collagen Matrices

Primary cultures of newborn human dermal fibroblasts (American Typeculture Collection) were seeded at a density of 1, 2 or 3×10⁵ cells perwell on 24-well flat bottom tissue culture plates either with or withoutmatrices (0.9 cm diameter) and grown for 4 hrs in Dulbeccos's modifiedeagle's medium (DMEM) supplemented with 10% fetal bovine serum,penicillin (100 units/ml), streptomycin (100/g/ml), 0.1 mM nonessentialamino acids and 2 mM 1-glutamine to determine cell attachment. Cellswere incubated at 37° C. with 5% CO₂ in dishes with and withoutmatrices. To measure cell growth with time, 3×10⁵ cells were seeded perwell and cultured for up to one week. Cell density was determined forthree plates per condition at the various times using the MTT assay (SeeMosmann, T., J. of Immunological Methods, 65:55-63 (1983)) Briefly,matrices were removed from the culture dishes prior to assaying. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) wasdissolved in phosphate-buffered saline (PBS) at 5 mg/ml and filtersterilized. Media of all wells to be assayed were replaced with 1 mlfresh media. An aliquot of 100 μl stock MTT solution was added to eachwell. The plates were incubated at 37° C. for 4 hours. After incubation,1 ml acid-isopropanol (0.04 N HCl in isopropanol) was added to eachwell. The solution was mixed by vortexing vigorously and the opticaldensity at 570 nm wavelength was measured using a spectrophotometer.

In general, fibroblasts seeded onto the collagen matrix attached morepoorly and proliferated more slowly than when seeded on plastic culturedishes. However, those cells which were grown on matrices crosslinkedusing the dehydrothermal technique allowed cell attachment and growth atlevels which approached those produced on tissue culture dishes alone.Preliminary experiments have demonstrated that the doubling time forfibroblasts grown on collagen matrices is increased from 18 hrs to 50hrs compared with fibroblasts grown on plastic (not shown). Similarresults have been reported for fibroblasts contained within LSE'sprepared from soluble collagen. In the case of LSE's, after the gelsundergo contraction, fibroblast proliferation was reduced to a steadyrate which was slower than that seen for cells grown on culture dishes.When human dermal fibroblasts were seeded onto sponges at a density of300,000 per well of 24 well plates, cell growth was similar for cellsgrown on the fibrillar collagen matrix compared with plastic. Cellgrowth on a hemostatic sponge (INSTAT) was lower than on plastic, andcell number decreased with time. The decrease in cell number may havebeen due to cell detachment, cell death or cell migration off of thesponge. The exact cause was not determined here.

Since fibroblasts proliferated slowly on the three-dimensional matrix,the number of fibroblasts introduced during the seeding had to beincreased in order to provide sufficient fibroblasts to form a dermis tosupport epidermal cell growth. Seeding at a concentration of 2.5×10⁵cells/cm² of matrix has been found to provide sufficient fibroblasts.

The presence of polylysine changed the surface charge of the matrix,improved the growth of fibroblasts and improved the ability of thematrices to support epidermal cell growth and differentiation.

EXAMPLE 4

Growth of Fibroblasts for In-vitro SMS Production

Matrices were soaked overnight in DMEM supplemented with 10% calf serumto remove residual acid. They were then seeded with 0.2-1×10⁶ humandermal fibroblasts per 2×2 cm sponge in the same media. Fibroblasts werecultured on the matrix for 7 days, and transferred to sterile petridishes and seeded with human epidermal cells as described below.

EXAMPLE 5

Preparation of Comparative Living Skin Equivalents LSE)

For purposes of comparison, LSE's were prepared according to the methodof Bell et al. See, Bell. E. B. et al., "Production of a tissue-likestructure by contraction of collagen lattices by human fibroblasts ofdifferent proliferative potential in vitro, " Proc. Natl. Acad. Sci.USA, 76:1274-1278 (1979). Primary human dermal fibroblasts weresuspended in DMEM containing 10% calf serum at 1.0-1.4×10⁵ cells/ml. Oneml of cell suspension and 2 ml of the same culture medium were added to1 ml of rat tail collagen (Type I; collaborative Research, Inc.) at aconcentration of 2.5 mg/ml in 10 mM acetic acid. The cellsuspension-collagen solution was poured into a 35 mm petri dish andincubated in a 37° C. tissue culture incubator in 5% CO₂ for 5 days toallow contraction to occur.

EXAMPLE 6

Effect of Matrix on Attachment and Growth of Cells

To determine the effect of different constructions and components ofcollagen matrices on the ability of cells to attached to the matrix,cells were applied to plastic tissue culture plates alone (PLASTIC) andto different collagen matrices as follows:

INSTAT--a commercially available collagen sponge

INSTAT--C commercially available collagen sponge crosslinked withcyanamide (crosslinked by treatment with 1% solution of cyanamide inneutral pH)

UMDNJ-C--collagen matrix of this invention crosslinked with cyanamide(crosslinked by treatment with 1% solution of cyanamide in neutral pH)

UMDNJ-DHT--collagen matrix of this invention crosslinked bydehydrothermal treatment

UMDNJ-DHT-PL--collagen matrix of this invention crosslinked bydehydrothermal treatment and incorporating polylysine

UMDNJ-DHT-PA--collagen matrix of this invention crosslinked bydehydrothermal treatment and incorporating polyaspartic acid

The cultures were assayed four hours later for cell density. The abilityof dermal fibroblasts to attach to these matrices is shown in FIG. 3.

Cyanamide crosslinking of collagen matrices inhibited the attachment offibroblasts to either a commercially available collagen sponge(INSTAT-C) or the matrix produced here (UMDNJ-C). Those matrices whichwere crosslinked using a dehydrothermal technique (UMDNJ-DHT) permittedcell attachment at levels which were closer to that seen with plastictissue culture plates alone (PLASTIC). The addition of polylysine to thematrix (UMDNJ-DHT-PL) had perhaps a slight enhancement of cellattachment while polyaspartic acid (UMDNJ-DHT-PA) had no effect.

To further analyze the ability of a collagen matrix to influence thegrowth of cultured cells, fibroblasts were grown on collagen matricesfor 1-7 days and assayed for cell density at various points along thistime course. In FIG. 4, the ability of cells to grow on plastic tissueculture dishes (PLASTIC) is compared to their ability to grow on thesame commercially available collagen sponge (INSTAT), a cyanamidecrosslinked version of the same matrix (INSTAT-CYANAMIDE), and acyanamide crosslinked version of the matrix described here(UMDNJ-CYANAMIDE). All of the matrices tested in this experimentdemonstrated reduced cell densities compared to tissue culture plastic;however, the UMDNJ matrix permitted better cell growth than thecommercial collagen sponge. Interestingly, although cyanamidecrosslinking was shown to inhibit cell attachment, it was shown here toenhance cell growth (compare INSTAT to INSTAT-CYANAMIDE).

The effect of added polyamino acids on cell growth in collagen matricesis shown in FIG. 5. No dramatic effect on cell growth was seen with theincorporation of either polylysine (UMDNJ-DHT-PL) or polyaspartic acidto matrices crosslinked by the dehydrothermal technique (UMDNJ-DHT).However, all of the matrices which were crosslinked by thedehydrothermal technique, either with or without additional molecules,performed nearly as well as cells grown on plastic culture dishes(PLASTIC) alone.

EXAMPLE 7

Keratinocyte Cultures

Human keratinocytes were obtained from foreskin (Clonetics Corporation)and grown for five days in Keratinocyte Basal Medium (KBM) supplementedwith Epidermal Growth Factor (EGF), insulin, hydrocortisone and BovinePituitary Extract (all from Clonetics) according to the proceduredescribed in Clonetics Technical Bulletin. Prior to use, the culturemedia were switched to an epidermal growth media (E-media) containing3:1 of high-glucose version of DMEM supplemented with Ham's F-12Nutrient Mixture (Gibco), followed by the addition of 5×10⁻¹⁰ M choleratoxin (Schwarz-Mann Biotech). In addition, 2 μg/ml hydrocortisone, 25μg/ml insulin, 25 μg/ml transferrin, and 1×10⁻¹⁰ M triiodothyronine (allfrom Sigma Chem. Co.) were added to prepare E-media.

EXAMPLE 8

Addition of Keratinocytes to Dermal Matrices

After fibroblast culture of five to seven days, 1.0×10⁶ keratinocyteswere added to the surface of the collagen matrix of Example 4 and to thesurface of the LSE of Example 5. The resulting cultures were grown at37° C. and 7% CO₂, submerged in E-media. Cultures remained submerged forapproximately one week to allow for epidermal attachment and monolayerformation. At this time they were placed on a steel mesh grid at theliquid-air interface. Cultures were then fed every other day withE-media. The dermal matrices were harvested 21 days (except where noted)after raising the culture to the liquid/air interface. The tissues werefrozen in liquid nitrogen until they were sectioned.

The skin model systems of this invention (based on collagen matrices asprepared in Example 1) were examined by light microscopy and compared tohuman neonatal foreskin. (FIGS. 6A-F) In normal skin (FIG. 6A), a dermallayer consisting of connective tissue and fibroblasts was apparent overwhich was a stratified epidermal layer containing keratinocytes ofvarying degrees of differentiation. The layers include a stratumbasalis, spinosum, granulosum and corneum. When keratinocytes werecultured on the Bell skin equivalent for 10 days (FIG. 6B), stratifiedlayers similar to those seen in foreskin were evident. In the stratumcorneum-like layer, nucleated cells persisted unlike that which occursin normal skin. Also evident was the lack of vasculature in thedermal-like layer which also contained fewer fibroblasts compared withskin. With longer term cultures (up to 21 days), these stratified layerspersisted (FIG. 6C) although the stratum corneum appearedhyperkeratinized.

When the keratinocytes were seeded and cultured on the air side ofcross-linked, insoluble collagen matrices (unmodified by polylysine), nodistinct layers were formed (FIG. 6D). Epidermal cells grew across thesurface of the matrix and within open spaces which were exposed to thesurface. Within these spaces were pockets of epidermal cells which grewin whorls in the interior of the matrix. These epidermal cells weredifferentiated in some respects as was evident by immunostaining (datanot shown).

With the incorporation of polylysine into the matrices and the additionof keratinocytes to the pan side of the matrix only, most sectionsshowed only one surface layer of epidermal cells, although there werealso pockets of epidermal cells near the surface of the matrix (FIG. 6E)seen in some of the outermost regions of the tissue only. Most areas ofthe matrices demonstrated morphologically distinct layers (FIG. 6F) asshown in the Bell skin equivalents (FIG. 6B). The stratum corneum-likelayer produced by epidermal cells cultured on the collagen matricescontaining polylysine was parakeratotic as was also demonstrated forepidermal cells cultured on skin equivalents.

EXAMPLE 9

Immunohistochemical Localization

In order to evaluate the level of differentiation of keratinocytes, bothculture systems were examined using antibodies to involucrin, a cellenvelope protein present in all but the basal layers of normal skin andone of the earlier proteins produced for the formation of the corneocyteenvelope in human skin; filaggrin, a protein produced in the suprabasallayer as profilaggrin and processed to filaggrin; keratin K1 which ispresent in the most highly differentiated keratinocytes; and loricrin, amajor cornified envelope precursor, one of the latest envelope proteinsto be expressed in normal skin.

Tissues were embedded in Histo Prep embedding compound (FisherScientific). Four micron sections of both foreskin and epidermalcultures were cut from frozen tissue on a cryostat (Minotome Corp.) andmounted on gelatin coated slides, air dried, and fixed in acetone (-20°C.) for five minutes. Sections were stained by standard indirectimmunochemical techniques. See, for example, Vadesande, F., et al.,"Peroxidase-antiperoxidase Techniques," in Methods in the Neurosciences,(Cuello, A. C., ed.), John Wiley & Sons: Chichester, Pa 3:101-120(1983).

The primary antibodies used were: mouse monoclonal anti-human filaggrin(Biomedical Technologies, Inc.) and rabbit anti-human involucrin (seeSimon, M. A., et al., "Involucrin in the epidermal cells ofsubprimates," J. Invest. Dermatol., 92:721-724 (1989)), rabbitanti-mouse K 1 (see Rosenthal, D. S., et al., "A human epidermaldifferentiation-specific keratin gene is regulated by calcium but notnegative modulators of differentiation in transgenic mousekeratinocytes," Cell Growth and Different., 2:107-113 (1991)), or rabbitanti-mouse loricrin (see Hohl, D., et al., J. Biol. Chem., 2666626-6636(1991)).

Control sections were incubated with the appropriate nonimmuneantiserum. Those sections visualized by immunofluorescence were hydratedin PBS (phosphate buffered saline) followed by sequential incubation in12% BSA (Sigma Chemical Co.) in 0.01M Tris-buffered saline (TBS), normalblocking serum, primary antibody, and finally the fluorescein-conjugatedsecondary antibody (Sigma Chemical Co.). For immunoenzyme staining, theprocedure was similar except the last incubation was replaced by asequential incubation in biotinylated secondary antibody (Vector)followed by the avidin-biotin-peroxidase complex (Vector). Antibodybinding was then visualized using 3,3'-diaminobenzidine (Sigma ChemicalCo.). The sections were then counterstained in Hematoxylin (SigmaChemical Co.) and mounted. All incubations were done in a humiditychamber.

Involucrin--The presence of involucrin was shown, by staining, in allbut the basal layers of the skin. Staining of the LSE (per Examples 5and 7) produced stained positive for involucrin in all suprabasallayers. When the skin model system (SMS) of this invention was examined,similar suprabasal staining was seen throughout the epidermis, however,light staining was seen in the basal layer. Some nonspecific dermalstaining of the collagen matrices was noted in both culture models.Sections incubated in nonimmune serum as a control showed no staining ofeither collagen matrix model.

Filaggrin--Filaggrin is found in human skin in the upper stratumspinosum and granulosum. Generally, staining of the stratum corneum doesnot indicate the presence of filaggrin, possibly because filaggrin isdegraded to amino acids in that layer. In LSE, the stratum granulosumand spinosum-like layers stained positively with no staining seen in thestratum corneum-like layer. Similar distribution of staining was seen inthe SMS developed here when stained for filaggrin. Control sectionsincubated with non-immune serum showed no staining.

Keratin K1--In normal skin, K1 staining was localized to the upperstratum spinosum and granular layers. In the SMS developed here, astaining pattern similar to that of foreskin was seen. With the LSE,light staining was present in a distribution similar to that of foreskinand the skin model system. Some staining was also seen in the stratumcorneum. Background dermal staining was present in both the skin modelsystem and foreskin. Sections incubated with nonimmune immunoglobulin asa control showed no staining.

Loricrin--When using an antibody specific to the epitopes on theN-terminal portion of loricrin, foreskin stained specifically from theupper stratum spinosum to the stratum corneum. When LSE cultures werestained with antibodies to loricrin, similar results were obtained. Withthe skin model system of this invention, a lighter but consistentstaining pattern was seen when compared to tissue equivalents. A similarpattern of staining and difference in intensity between models was seenwith an antibody directed at the C-terminal portion of loricrin.Non-immune serum controls showed no staining.

The foregoing examples demonstrate the ability of a crosslinked collagenmatrix to serve as a substrate for the assembly of an in vitro skinmodel system which compares favorably to the model produced by themethods of Bell et al. and normal skin. This model possesses a number ofattributes which represent potential advantages over currently availabletechnologies of this kind. The skin model system of this invention isless expensive to manufacture than the living skin equivalent utilizingsoluble collagen and living cells. Since the base collagen matrix ismade without living cells, it is possible to modify the matrix or tocovalently or noncovalently incorporate additional components into thematrix by a process which could be detrimental to living cells (e.g.,the dehydrothermal crosslinking step). The collagen matrix of the skinmodel system of this invention can be stored in its lyophilized state.In contrast, the collagen matrix of the living skin equivalent containsliving cells and must be maintained under special conditions. The skinmodel system of this invention can be made in a wide variety of shapesand sizes and is noncontracting under normal conditions. Thecollagen/cell matrix of the LSE contracts as the cells proliferate,making it difficult to produce a sheet to the desired size and shape.

What is claimed is:
 1. A method for preparing a skin model systemcomprising(a) providing a three-dimensional, cross-linked porous matrixof insoluble collagen, (b) seeding said matrix with fibroblasts andculturing the seeded matrix under conditions to allow ingrowth andproliferation of said fibroblasts, (c) seeding the surface of theproduct of step (b) with epidermal cells in a manner to deter ingrowthof said epidermal cells, (d) culturing the product of step (c) for afirst period of time under conditions to allow said epidermal cells toattach to said matrix and proliferate to form a monolayer, and (e)culturing the product of step (d) for a second period of time underconditions to allow said epidermal cells to differentiate.
 2. The methodof claim 1 wherein said three-dimensional, cross-linked porous matrixhas two major surfaces opposed to one another, one of said majorsurfaces having a substantially smaller average pore size than theother.
 3. The method of claim 2 wherein the average pore size of one ofsaid major surfaces of said matrix is at least about 100μ smaller thanthe average pore size of said other major surface.
 4. The method ofclaim 2 wherein said epidermal cells are seeded onto said major surfacehaving a substantially smaller average pore structure.
 5. The method ofclaim 2 wherein in step (d) said matrix is submerged in an epidermalgrowth media.
 6. The method of claim 1 wherein in step (e) said matrixis held in epidermal growth media so that the epidermal-cell containingsurface matrix of said matrix is exposed to the atmosphere.
 7. A methodof claim 1 wherein said three-dimensional, cross-linked porous matrix ofinsoluble collagen is prepared by a method comprising(a) placing adispersion of insoluble collagen in a suitable mold tray, (b) subjectingsaid dispersion to freezing and lyophilization conditions, (c)subjecting the product of step (b) to cross-linking conditions toproduce said three-dimensional, cross-linked porous matrix.
 8. A methodof claim 7 wherein said crosslinking conditions comprise exposure totemperatures of from about 50° C. to about 200° C. at a vacuum of50millitorr or less.
 9. A method for treating a patient suffering from awound to the skin comprising the step of:applying to said wound the skinmodel system comprising a three-dimensional cross-linked porous matrixof insoluble collagen containing fibroblast cells therein, andstratified layers of differentiated epidermal cells supported thereon.10. A method for preparing a skin model system comprising:(a) forming athree-dimensional, cross-linked porous matrix of insoluble collagen in amold tray, wherein said matrix has a mold surface that is in directcontact with said mold tray at time of formation of said matrix in saidmold tray, and an air surface, (b) seeding said matrix with fibroblastsand culturing said matrix under conditions to allow ingrowth andproliferation of said fibroblasts, (c) seeding said mold surface withepidermal cells, (d) culturing the product of step (c) for a firstperiod of time under conditions to allow said epidermal cells to attachto said mold surface and proliferate to form an epidermal-cellcontaining surface, (e) culturing the product of step (d) for a secondperiod of time under conditions to allow said epidermal cells todifferentiate.
 11. The method of claim 7 wherein in step (b) saidfibroblasts are seeded and cultured on said air surface.
 12. The methodof claim 7 wherein in step (d) said matrix is submerged in an epidermalgrowth media.
 13. The method of claim 7 wherein in step (e) said matrixis held in epidermal growth media so that the mold surface is exposed tothe atmosphere.
 14. The method of claim 7 wherein saidthree-dimensional, cross-linked porous matrix of insoluble collagen isprepared by a method comprising:(a) placing a dispersion of insolublecollagen in said mold tray, (b) subjecting said dispersion to freezingand lyophilization conditions, (c) subjecting the product of step (b) tocross-linking conditions to produce said three-dimensional, cross-linkedporous matrix.
 15. A method for preparing a skin model systemcomprising:(a) seeding a three-dimensional, cross-linked porous matrixof insoluble collagen that is found in a mold tray with fibroblastswherein said matrix has a mold surface and an air surface, (b) seedingsaid mold surface with epidermal cells, (c) culturing the product ofstep (b) for a first period of time under conditions to allow saidepidermal cells to attach to said mold surface and proliferate tosubstantially envelop said mold surface, (d) culturing the product ofstep (c) for a second period of time under conditions to allow saidepidermal cells to differentiate.
 16. The method of claim 10 wherein instep (b) said fibroblasts are seeded and cultured on said air surface.17. The method of claim 10 wherein in step (d) said matrix is submergedin an epidermal growth media.
 18. The method of claim 10 wherein in step(e) said matrix is held in epidermal growth media so that said moldsurface is exposed to the atmosphere.
 19. The method of claim 10 whereinsaid three-dimensional, cross-linked porous matrix of insoluble collagenis prepared by a method comprising:(a) placing a dispersion of insolublecollagen in said mold tray, (b) subjecting said dispersion to freezingand lyophilization conditions, (c) subjecting the product of step (b) tocross-linking conditions to produce said three-dimensional, cross-linkedporous matrix.
 20. A method for treating a patient suffering from awound to the skin comprising applying to said wound a skin model system,wherein said skin model system comprises:(a) a three-dimensional,cross-linked, porous matrix of insoluble collagen containing fibroblastcells therein, (b) stratified layers of differentiated epidermal cellssupported thereon, wherein a layer of said differentiated epidermalcells is in direct contact with a surface of said three-dimensional,cross-linked, porous matrix of insoluble collagen.
 21. The method ofclaim 20 wherein said differentiated epidermal cells comprisekeratinocytes.
 22. The method of claim 21 wherein said fibroblasts aredermal fibroblasts.
 23. The method of claim 20 wherein said collagenmatrix has been cross-linked by a dehydrothermal method.
 24. The methodof claim 20 wherein said skin model system is in the form of a sheethaving a thickness of in the range of about 1 to 5 mm.
 25. A method fortreating a patient suffering from a wound to the skin comprisingapplying to said wound a skin model system comprising:(a) athree-dimensional, cross-linked, porous matrix of insoluble collagencontaining fibroblast cells therein, (b) a monolayer of differentiatedepidermal cells supported thereon in direct contact with a surface ofsaid three-dimensional, cross-linked, porous matrix of insolublecollagen.
 26. The method of claim 25 wherein said differentiatedepidermal cells comprise keratinocytes.
 27. The method of claim 26wherein said fibroblasts are dermal fibroblasts.
 28. The method of claim25 wherein said collagen matrix has been cross-linked by adehydrothermal method.
 29. The method of claim 25 wherein said skinmodel system of claim 6 is in the form of a sheet having a thickness ofin the range of about 1 to 5 mm.